Expression and characterization of human single chain Fv antibody to anti-glomerular basement membrane (anti-GBM) antibody in Escherichia coli

Objective:To construct the prokaryotic expressing plasmid for human scFv3 gene against or of anti-GBM antibody and prepare the scFv3 protein for identifying biological function.Methods:The scFv3 gene of anti-GBM antibody was amplified by PCR and cloned into pGEM-T Easy Vector.The sequence of cloned scFv3 was confirmed by restriction analysis and DNA sequencing.A scFv3 prokaryotic expressing plasmid,pQE80L-scFv3,was then constructed by subclone.pQE80L-scFv3 transformed E.coli Rosetta2 was induced by IPTG.The expression of scFv3 protein was analyzed by SDS-PAGE and Western blot.Results:The amplified DNA fragment was in size of 750 bp as expected.Restriction analysis showed that the amplified gene was inserted in pQE80L correctly.Sequence showed there was no mutation in both ends of the cloned scFv3.Restrict analysis showed that the recombinant pQE80L-scFv3 was right.A Mr 27×103 protein could be seen in Rosetta2 induced by IPTG with SDS-PAGE and was positive while reacting with anti-GBM antibody by Western blot.Conclusion:The construction of the prokaryotic expression plasmid of scFv3 gene and preparation of scFv3 fragment establish a solid basis of further studying the biological function of the human scFv3 to anti-GBM antibody.